Project summary The tears, for the most part, have an electrolyte composition like other extracellular fluids. The tears are unique, however, in that the K+ concentration ([K+] is about 20-25 mM, compared to about 5 mM in other extracellular fluids. Exposure to ultraviolet radiation causes apoptosis of cells. UVB radiation at doses relevant to ambient levels of outdoor UVB exposure activates K+ channels leading to rapid loss of intracellular K+ (K+i). We have shown that incubation of corneal epithelial cells in medium with 25 mM K+ after exposure to UVB prevents loss of K+i and inhibits activation of apoptotic mechanisms. These observations support our overall hypothesis that the high concentration of potassium ions in tears contributes to the protection of the corneal epithelium from ambient UVB-induced damage by reducing the loss of intracellular K+ when channels are activated by UVB. We now propose to investigate the intracellular signaling mechanisms by which UVB activates K+ channels and apoptotic signaling in corneal epithelial cells. The specific aims of the proposed research are: 1) to elucidate the relationships between Fas ligand- independent, UVB-induced activation of apoptotic signaling pathways and activation of K+ channels. This will be accomplished by studying the effects of UVB on apoptotic pathways and K+ channel activation in corneal epithelial cells after siRNA knockdown of Fas and FADD, or inhibition of caspase-8; 2) To investigate whether UVB-induced K+ channel activation is the primary event in activation of apoptotic pathways. K+ channels in corneal epithelial cells will be identified using a PCR array. siRNA will then be used to knock down specific channels. This will be followed by investigation of UVB-induced activation of apoptosis, as well as effects of UVB on K+ currents and intracellular [K+] when specific channels have been knocked down; 3) Using an isolated pig eye model, effects of UVB on the intact corneal epithelium will be investigated in a configuration that allows for superfusion of the ocular surface with physiologic saline in a manner that models the bathing of the cornea with tears. The hypothesis will be tested that elevated [K+] will protect the superficial corneal cell layers from UV-induced apoptosis.